Materials and methods for treatment of allergic diseases

ABSTRACT

The present invention pertains to a method for treatment of allergic diseases by administering a natriuretic hormone peptide (NHP), or a nucleic acid sequence encoding NHP, to a patient in need thereof. In another aspect, the present invention concerns an expression vector comprising a nucleic acid sequence encoding NHP. In another aspect, the present invention concerns a host cell genetically modified with a nucleic acid sequence encoding NHP. In another aspect, the present invention concerns a pharmaceutical composition comprising NHP or a nucleic acid sequence encoding NHP and a pharmaceutically acceptable carrier. In another aspect, the present invention pertains to novel fragments of atrial natriuretic peptide (ANP) exhibiting bronchodilatory and anti-inflammatory activity, and isolated nucleic acid sequences encoding the fragments.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a National Stage filing of International ApplicationNumber PCT/US2003/028056, filed Sep. 8, 2003, which claims the benefitof U.S. Provisional Application No. 60/319,529, filed Sep. 6, 2002,which is hereby incorporated by reference herein in its entirety,including any figures, tables, nucleic acid sequences, amino acidsequences, or drawings.

BACKGROUND OF THE INVENTION

Respiratory diseases, such as allergic rhinitis, asthma, and chronicobstructive pulmonary disorders (COPD) are often debilitating conditionswith high prevalence, affecting more than 155 million people in thedeveloped world. Asthma is one of the common chronic diseases and israpidly increasing by 20% to 50% per decade, particularly in children.Currently, there are 53 million patients in the major pharmaceuticalmarkets. Constriction of the airways is the hallmark of chronicconditions such as asthma and COPD, and inflammation is common to allrespiratory diseases affecting either the upper or lower airways.Bronchodilators, which may possess limited anti-inflammatory activity,are considered the first line of therapy for asthma. Steroids areconsidered the gold standard as anti-inflammatory therapy, but theypossess other significant adverse effects. Effective therapeutics otherthan steroids are under intense investigation.

A group of four peptide hormones, originating from the 126-amino acidatrial natriuretic factor (ANF) prohormone, have been known for theirvasodilator activity. These four peptide hormones, consisting of aminoacids 1-30, 31-67, 79-98, and 99-126 of this prehormone, have been namedlong acting natriuretic peptide, vessel dilator, kaliuretic peptide, andatrial natriuretic peptide (ANP), respectively for their most prominenteffects (Angus R. M. et al., Clin Exp Allergy 1994, 24:784-788). The ANPsequence, particularly the C-terminal portion, is highly conserved amongspecies (Seidman et al., Science, 1984, 226:1206-1209). It has beenproposed to be useful for treatment of various cardiovascular,respiratory, cancerous and renal diseases (Vesely, D. L. Cardiovascular,2001, 51:647-658).

The C-terminal peptide of proANF (also known by the synonym proANP), ANPis a 28-amino acid hormone secreted by the cardiac atria and lung tissueNeedleham, P. et al., N Engl J Med, 1986, 314:828-834). ANP hasvasodilator, natriuretic and diuretic properties (Needleham, P. et al.,N Engl J Med, 1986, 314:828-834). ANP infused at high concentrationsreduces airway resistance in normal subjects (Hulks G. et al., Clin Sci1990;79:51-55) and produces a significant bronchodilator response inpatients with asthma. Inhaled ANP attenuates histamine- and methacholine(MCh)-induced bronchoconstriction (Hulks, G. et al., Br. Med J, 1992,304:1156; Angus, R. M. et al., Clin Exp Allergy, 1994, 24:784-788);however, the amount of ANP required for efficacy and their shorthalf-life limits their use for long-term modulation of airwayhyper-responsiveness (Hamet, P. et al., Nephrologie, 1987, 8:7-12;Matsuse, H., et al., J Immunol, 2000, 164:6583-6582).

The present inventor has demonstrated prolonged amelioration of symptomsassociated with respiratory allergy and asthma by delivery ofpDNA-encoding various natriuretic hormone peptides (NHPs), or bydelivery of the peptides themselves, which exhibit bronchodilatoryand/or anti-inflammatory activity.

BRIEF SUMMARY OF THE INVENTION

The present invention pertains to a method for treating respiratoryallergies, such as allergic rhinitis and asthma, which may be caused byallergens and exacerbated by respiratory viral infections, pollutants,and smoke.

In one embodiment, the method of the present invention comprisesadministering a therapeutically effective amount of a natriuretichormone peptide (referred to herein as NHP or NHP peptide) to a patientin need of such treatment. As used herein, NHP refers to atrialnatriuretic factor (ANF) hormone, or a biologically active fragment orhomolog thereof.

Specifically exemplified NHPs comprise an amino acid sequence selectedfrom the group consisting of amino acids 1-30 of ANF (also known as“long acting natriuretic peptide” and referred to herein as NHP₁₋₃₀ orSEQ ID NO:1), amino acids 31-67 of ANF (also known as “vessel dilator”and referred to herein as NHP₃₁₋₆₇ or SEQ ID NO:2), amino acids 79-98 ofANF (also known as “kaliuretic peptide” and referred to herein asNHP₇₉₋₉₈ or SEQ ID NO:3), and amino acids 99-126 of ANF (also known as“atrial natriuretic peptide” or “ANP”, and referred to herein asNHP₉₉₋₁₂₆ or SEQ ID NO:4), or biologically active fragments or homologsof any of the foregoing. Other exemplified NHPs comprise amino acids73-102 of proANF (referred to herein as NHP₇₃₋₁₀₂ or SEQ ID NO:5), orSEQ ID NO:6, or biologically active fragment(s) or homolog(s) of theforegoing. In one embodiment, the NHP administered to the patient doesnot consist of NHP₉₉₋₁₂₆ (SEQ ID NO:4).

In another embodiment, the method of the present invention comprisesadministering an effective amount of at least one nucleic acid moleculeencoding an NHP to a patient in need of such treatment. The presentinventor has determined that introduction of a nucleic acid moleculeencoding NHP is capable of inhibiting allergen-specific IgE synthesisfor the treatment of allergic disease. The gene delivery method of thepresent invention permits long-term expression of NHP-encoding nucleicacid sequences in vivo, thereby conferring bronchoprotective effectand/or anti-inflammatory effect against respiratory allergies, such asasthma. In one embodiment, a therapeutically effective amount of atleast one nucleic acid molecule encoding a peptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 orbiologically active fragments or homologs of any of the foregoing, areadministered to the patient.

In another aspect, the present invention concerns an isolated peptidecomprising the amino acid sequence NHP₇₃₋₁₀₂ (SEQ ID NO:5) or SEQ IDNO:6, or a biologically active fragment or homolog of the foregoing. Inanother aspect, the present invention concerns an isolated nucleic acidmolecule encoding the amino acid sequence of NHP₇₃₋₁₀₂ (SEQ ID NO:5) orencoding the amino acid sequence of SEQ ID NO:6, or a biologicallyactive fragment or homolog thereof.

In another aspect, the present invention concerns an expression vectorcomprising a nucleic acid sequence encoding an NHP, and a promotersequence that is operably linked to the NHP-encoding nucleic acidsequence. In one embodiment, the expression vector is a DNA plasmid orvirus. In another aspect, the present invention concerns apharmaceutical composition comprising a nucleic acid sequence encodingan NHP, and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and objects of the invention,reference should be made to the following detailed description, taken inconnection with the accompanying drawings, in which:

FIG. 1 shows a diagram depicting the family of natriuretic hormonepeptides (NHP). Translation of the atrial natriuretic factor generesults in a pre-prohormone from which the 5′ signal sequence is cleavedto yield the 126 amino acid prohormone (ANF). The prohormone is furthercleaved by endopeptidases into several bioactive peptides, long-actingnatriuretic peptide (LANP) (NHP₁₋₃₀; SEQ ID NO:1), vessel dialator (VD)(NHP₃₁₋₆₇; SEQ ID NO:2), kaliuretic peptide (KP) (NHP₇₉₋₉₈; SEQ IDNO:3),and atrial natriuretic peptide (ANP) (NHP₉₉₋₁₂₆; SEQ ID NO:4).Urodialatin (UD), a variant of ANP is also in circulation and has beenimplicated in asthma. The NHP construct used in these studies encodesNHP₇₃₋₁₀₂ (SEQ ID NO:5) and SEQ ID NO:6, which are distinct from theabove peptides but include a critical overlap region shared with UD andANP.

FIGS. 2A and 2B show cloning and expression of NHP in human alveolarcells and effect on AHR of murine lung. FIG. 2A shows the successfulcloning of the peptides of ANF in the pVAX expression vector. The gelelectrophoresis of excised inserts corresponding to SEQ ID NO:13(approximately 114 bp band) and to NHP₇₃₋₁₀₂ (approximately 90 bp band)are shown. FIG. 2B shows expression of NHP₇₃₋₁₀₂ in human Type IIalveolar epithelial cells, A549. A549 cells were either transfected withpNHP₇₃₋₁₀₂, SEQ ID NO:13 or pVAX vector control. Expression of ANP-likepeptide was detected in cell supernatant and lysate from transfectedcells alone but not pVAX control. *p<0.05; compared to pVAX control.

FIGS. 3A-3E show therapeutic effect of NHP₇₃₋₁₀₂ on asthma in mice. FIG.3A shows an experimental outline of immunization protocol usingNHP₇₃₋₁₀₂. FIG. 3B shows expression of NHP₇₃₋₁₀₂ in murine lung. Micewere administered intranasally (i.n.) either with pNHP₇₃₋₁₀₂ or pVAX asdescribed. Three days following the last DNA administration, NHPexpression was checked from lung tissue by RT-PCR. Mice receivingNHP₇₃₋₁₀₂ (lane 2) exhibited NHP expression, which was not present incontrol mice receiving empty pVAX plasmid (lane 1). FIG. 3C shows anestimation of the degree of sensitization following ova injection (FIG.3A). Mice (n=4) were injected intraperitonealy (i.p.) either with ovaand alum or phosphate buffered saline (PB), and on day 21 their serumwas analyzed for ova specific IgE. Mice receiving ova and alum exhibitedhigher titers (p<0.01) of ova specific IgE than the PBS control. Theexperiments were repeated twice and data from a representativeexperiment are shown. FIGS. 3D-E show the measurement of AHR toincreasing concentrations of methacholine following NHP gene transfer onday 26. BALB/c mice (n=4) were sensitized with ovalbumin by i.p.immunization (10 μg/mouse) and 14 days later were treated with 10μg/mouse of SEQ ID NO: 13 or pNHP₇₃₋₁₀₂ itranasally. The control groupreceived the empty vector alone. Each mouse was intranasaly administeredthree times on two days interval with 10 μg of plasmid DNA complexedwith 50 μg of transfection reagent Lipofectamine (Life Technologies,Rockville, Md.). Animals were challenged with the same allergen (50 μgin PBS) three days after the last intransal DNA delivery and 24 hourslater their AHR was measured using the whole body plethysmograph (Buxco,Troy, N.Y.). A dose-dependent decrease of methacholine response is shownin FIG. 3D. FIG. 3E shows the effect of treatment with SEQ ID NO:13 andpNHP₇₃₋₁₀₂ on allergen-induced airway hyper-responsiveness (AHR). Theeffect of treatment at the highest concentration (50 mg/ml) ofmethacholine challenge is shown (p<0.05).

FIGS. 4A and 4B show the long-term effect on AHR following prophylaxisby NHP₇₃₋₁₀₂ gene transfer. FIG. 4A shows schematically the protocol ofsensitization, treatment and antigen challenges and measurement of AHR.FIG. 4B shows measurement of Penh (%) at 50 mg/ml of methachohne.*p<0.05; compared to pVAX control. The experiment was repeated twice anddata from a representative experiment are shown.

FIGS. 5A-5C show that administration of chitosan-pNHP nanoparticlesexhibit a therapeutic effect for allergen-RSV induced asthma andreversal of asthma in mice. FIG. 5A shows an experimental outline ofimmunization protocol with allergen and RSV, treatment schedules,challenges and AHR measurements. FIG. 5B shows reversal of airwayhyper-reactivity as evident from % Penh measurement following treatmentwith chitosan+pNHP₇₃₋₁₀₂. The other treatments include chitosan+pVAX(control), chitosan+NHP₇₃₋₁₀₂, fluticasone, and fluticasone andsalmeterol alone. FIG. 5C shows the reduction in inflammatory cells inthe lung by treatment with pNHP₇₃₋₁₀₂. Mice treated as shown in FIG. 5Bwere subjected to bronchioalveolar lavage (BAL) following AHR. A BALcell differential was performed and cytospun BAL cells were stained anddifferent cell types were quantified by three blinded investigators. Thepercentage of cells of macrophages, eosinophils, neutrophils andlymphocytes were determined.

FIGS. 6A-6C show that overexpression of NHP₇₃₋₁₀₂ leads to increasedproduction of nitric oxide in human epithelial cells. A549 (FIG. 6A) andNHBE (FIG. 6B) cells were transfected with control vector or NHP₇₃₋₁₀₂.At the indicated times after transfection, aliquots of the culturemedium were assayed for nitrite (the NO reaction product). Fluorescencewas read at 409 nm with excitation at 365 nm using a JASCOspectrofluorometer. Data are means±SEM (n=3). FIG. 6C shows that NOproduction is due to the constitutive NOS. One aliquot of cells wasincubated during the expression phase with 1 mM N_(ω)-nitro-L-argininemethyl ester, an arginine analog that blocks cNOS production of NO(NHP+i). The enhanced NO generation was inhibited by pretreatment of thecells with N-nitro-L-arginine methyl ester, which blocks cNOS activity.

FIGS. 7A and 7B show that pNHP₇₃₋₁₀₂ exerts its anti-inflammatoryactivity in the lung by decreasing NFκB activation in epithelial cells.A549 (FIG. 7A) or NHBE (FIG. 7B) cells were co-transfected withpNHP₇₃₋₁₀₂ or vector pVAX (pV) alone, NFκB plasmid carrying theluciferase reporter gene pNFκB-luc reporter plasmid) (MERCURY PROFILINGSYSTEM, CLONTECH), and pLacZ normalization control. NFκB was activated24 hr after transfection by incubating cells with 20 ng/ml phorbolmyristoyl acetate (PMA) (for A549 cells) or 10 ng/ml of TNF-α (for NHBEcells). Luciferase activity was detected using the DUAL LUCIFERASEREPORTER Assay kit (CLONTECH) and DYNEX MLX luminometer. Data (averageof three readings±SEM) are expressed as fold change in luciferaseactivity in arbitrary units relative to vector control.

BRIEF DESCRIPTION OF THE SEQUENCES

-   SEQ ID NO:1 is the amino acid sequence of human “long acting    natriuretic peptide” or NHP₁₋₃₀: ¹NPMYN AVSNADLMDF KNLLDHLEEK    MPLED³⁰.-   SEQ ID NO:2 is the amino acid sequence of human “vessel dilator” or    NHP₃₁₋₆₇: ³¹EVVPP QVLSEPNEEA GAALSPLPEV PPWTGEVSPA QR⁶⁷.-   SEQ ID NO:3 is the amino acid sequence of human “kaliuretic peptide”    or NHP₇₉₋₉₈: ⁷⁹SSDRSAL LKSKLRALLT APR⁹⁸.-   SEQ ID NO:4 is the amino acid sequence of human “atrial natriuretic    peptide” (ANP) or NHP₉₉₋₁₂₆: ⁹⁹SLRRSSC FGGRMDRIGA QSGLGCNSFR Y¹²⁶.-   SEQ ID NO:5 is the amino acid sequence of cloned mouse pNHP₇₃₋₁₀₂:    ⁷³GSPWDPSDRSALLKSKLRALLAGPRSLRRS¹⁰² .-   SEQ ID NO:6 is the amino acid sequence of cloned mouse NHP fragment:    VSNTDLMDFKNLLDHLEEKMPVEDEVMPPQALSEQTE.-   SEQ ID NO:7 is the amino acid sequence for the human preproANP (NCBI    ACCESSION # NM_(—)006172) wherein the underlined amino acids    represent the signal sequence which is cleaved off to form the    mature peptide:

¹MSSFSTTTVS FLLLLAFQLL GQTRANPMYN AVSNADLMDF KNLLDHLEEK MPLEDEVVPPQVLSEPNEEA GAALSPLPEV PPWTGEVSPA QRDGGALGRG PWDSSDRSAL LKSKLRALLTAPRSLRRSSC FGGRMDRIGA QSGLGCNSFR Y¹⁵¹.

-   SEQ ID NO:8 is a forward primer for the cDNA sequence encoding mouse    prepro ANF protein:

5′-gac ggc aag ctt act atg ggc agc ccc tgg gac cc-3′.

-   SEQ ID NO:9 is a reverse primer for the cDNA sequence encoding mouse    pre-proANF protein:

5′-acc ccc ctc gag tta tta tct tcg tag gct ccg-3′.

-   SEQ ID NO:10 is a forward primer for the cDNA sequence encoding    mouse NHP fragment:

5′-aat cct aag ctt agt atg gtg tcc aac aca gat-3′

-   SEQ ID NO:11 is a reverse primer for the cDNA sequence encoding    mouse NHP fragment:

5′-tgc gaa ctc gag tta ctc agt ctg ctc act cag ggc ctg cg-3′

-   SEQ ID NO:12 is the nucleotide sequence encoding cloned mouse    pNHP₇₃₋₁₀₂:

atg ggc agc ccc tgg gac ccc tcc gat aga tct gcc ctc ttg aaa agc aaa ctgagg gct ctg ctc gct ggc cct cgg agc cta cga aga taa

-   SEQ ID NO:13 is the nucleotide sequence encoding cloned mouse pNHP    fragment:

atg gtg tcc aac aca gat ctg atg gat ttc aag aac ctg cta gac cac ctg gaggag aag atg ccg gta gaa gat gag gtc atg ccc ccg cag gcc ctg agt gag cagact gag taa

-   SEQ ID NO:14 is the mRNA nucleotide sequence encoding human ANP    (NCBI Accession # NM_(—)006172:

  1 tggcgaggga cagacgtagg ccaagagagg ggaaccagag aggaaccaga ggggagagac 61 agagcagcaa gcagtggatt gctccttgac gacgccagca tgagctcctt ctccaccacc121 accgtgagct tcctcctttt actggcattc cagctcctag gtcagaccag agctaatccc181 atgtacaatg ccgtgtccaa cgcagacctg atggatttca agaatttgct ggaccatttg241 gaagaaaaga tgcctttaga agatgaggtc gtgcccccac aagtgctcag tgagccgaat301 gaagaagcgg gggctgctct cagccccctc cctgaggtgc ctccctggac cggggaagtc361 agcccagccc agagagatgg aggtgccctc gggcggggcc cctgggactc ctctgatcga421 tctgccctcc taaaaagcaa gctgagggcg ctgctcactg cccctcggag cctgcggaga481 tccagctgct tcgggggcag gatggacagg attggagccc agagcggact gggctgtaac541 agcttccggt actgaagata acagccaggg aggacaagca gggctgggcc tagggacaga601 ctgcaagagg ctcctgtccc ctggggtctc tgctgcattt gtgtcatctt gttgccatgg661 agttgtgatc atcccatcta agctgcagct tcctgtcaac acttctcaca tcttatgcta721 actgtagata aagtggtttg atggtgactt cctcgcctct cccaccccat gcattaaatt781 ttaaggtaga acctcacctg ttactgaaag tggtttgaaa gtgaataaac ttcagcacca841 tggac

-   SEQ ID NO:15 is the human gene for atrial natriuretic factor    propeptide (coding sequence includes−join (570 . . . 692, 815 . . .    1141, 2235 . . . 2240); sig. peptide=570 . . . 644; mat.    peptide=join (645 . . . 692, 815 . . . 1141, 2235 . . . 2237), (NCBI    ACCESSION NO: X01471; Greenberg, B. D. et al., Nature, 1984,    312(5995):656-658):

   1 ggatccattt gtctcgggct gctggctgcc tgccatttcc tcctctccac ccttatttgg  61 aggccctgac agctgagcca caaacaaacc aggggagctg ggcaccagca agcgtcaccc 121 tctgtttccc cgcacggtac cagcgtcgag gagaaagaat cctgaggcac ggcggtgaga 181 taaccaagga ctctttttta ctcttctcac acctttgaag tgggagcctc ttgagtcaaa 241 tcagtaagaa tgcggctctt gcagctgagg gtctgggggg ctgttggggc tgcccaaggc 301 agagaggggc tgtgacaagc cctgcggatg ataactttaa aagggcatct cctgctggct 361 tctcacttgg cagctttatc actgcaagtg acagaatggg gagggttctg tctctcctgc 421 gtgcttggag agctgggggg ctataaaaag aggcggcact gggcagctgg gagacaggga 481 cagacgtagg ccaagagagg ggaaccagag aggaaccaga ggggagagac agagcagcaa 541 gcagtggatt gctccttgac gacgccagca tgagctcctt ctccaccacc accgtgagct 601 tcctcctttt actggcattc cagctcctag gtcagaccag agctaatccc atgtacaatg 661 ccgtgtccaa cgcagacctg atggatttca aggtagggcc aggaaagcgg gtgcagtctg 721 gggccagggg gctttctgat gctgtgctca ctcctcttga tttcctccaa gtcagtgagg 781 tttatccctt tccctgtatt ttccttttct aaagaatttg ctggaccatt tggaagaaaa 841 gatgccttta gaagatgagg tcgtgccccc acaagtgctc agtgagccga atgaagaagc 901 gggggctgct ctcagccccc tccctgaggt gcctccctgg accggggaag tcagcccagc 961 ccagagagat ggaggtgccc tcgggcgggg cccctgggac tcctctgatc gatctgccct1021 cctaaaaagc aagctgaggg cgctgctcac tgcccctcgg agcctgcgga gatccagctg1081 cttcgggggc aggatggaca ggattggagc ccagagcgga ctgggctgta acagcttccg1141 ggtaagagga actggggatg gaaatgggat gggatggaca ctactgggag acaccttcag1201 caggaaaggg accaatgcag aagctcattc cctctcaagt ttctgcccca acacccagag1261 tgccccatgg gtgtcaggac atgccatcta ttgtccttag ctagtctgct gagaaaatgc1321 ttaaaaaaaa aagggggggg gctgggcacg gtcgtcacgc ctgtaatccc agcactttgg1381 gaggccaggc agcggatcat gaggtcaaga gatcaagact atcctggcca acatggtgaa1441 accccagctc tactaaaaat acaaaaatta gctgggtgtg tggcgggcac ctgtactctc1501 agctacttgg gaggctgagg caggagaatc acttgaaccc aggaggcaga ggttgcagtg1561 agcagagatc acgccactgc agtccagcct aggtgataga gcgagactgt ctcaaaaaaa1621 aaaaaaaaag gccaggcgcg gtggctcacg cctgtaatcc cagcgctttg ggaggccaag1681 gcgggtggat cacgaggtca ggagatggag accatcctgg ctaacacggt gaaaccccgt1741 ctctactaaa aatacaaaaa attagccagg cgtggtggca ggcgcctgta agtcctagct1801 actccggagg ctgaggcagg agaatggcgt gaacccggga ggcggagctt gcagtgagca1861 gagatggcac cactgcactc cagcctgggc gacagagcaa gactccgtct caaaaaaaaa1921 aaaaaaaaaa gcaactgcca ctagcactgg gaaattaaaa tattcataga gccaagttat1981 ctttgcatgg ctgattagca gttcatattc ctccccagaa ttgcaagatc ctgaagggct2041 taagtgaaat ttactctgat gagtaacttg cttatcaatt catgaagctc agagggtcat2101 caggctgggg tgggggccgg tgggaagcag gtggtcagta atcaagttca gaggatgggc2161 acactcatac atgaagctga cttttccagg acagccaggt caccaagcca gatatgtctg2221 tgttctcttt gcagtactga agataacagc cagggaggac aagcagggct gggcctaggg2281 acagactgca agaggctcct gtcccctggg gtctctgctg catttgtgtc atcttgttgc2341 catggagttg tgatcatccc atctaagctg cagcttcctg tcaacacttc tcacatctta2401 tgctaactgt agataaagtg gtttgatggt gacttcctcg cctctcccac cccatgcatt2461 aaattttaag gtagaacctc acctgttact gaaagtggtt tgaaagtgaa taaacttcag2521 caccatggac agaagacaaa tgcctgcgtt ggtgtgcttt ctttcttctt gggaagagaa2581 ttc

-   SEQ ID NO:16 is the amino acid sequence for the mouse preproANP    peptide 1 mgsfsitlgf flvlafwlpg higanpvysa vsntdhndfk nlldhleekm    pvedevmppq 61 alseqteeag aalsslpevp pwtgevnppl rdgsalgrsp wdpsdrsall    ksklrallag 121 prslrrsscf ggridrigaq sglgcnsfry rr-   SEQ ID NO: 17 is the genetic sequence for the mouse preproANP    peptide wherein the coding sequence starts at nucleic acid molecule    position 81 and ends at nucleic acid molecule position 539.

  1 caaaagctga gagagagaga gaaagaaacc agagtgggca gagacagcaa acatcagatc 61 gtgccccgac ccacgccagc atgggctcct tctccatcac cctgggcttc ttcctcgtct121 tggccttttg gcttccaggc catattggag caaatcctgt gtacagtgcg gtgtccaaca181 cagatctgat ggatttcaag aacctgctag accacctgga ggagaagatg ccggtagaag241 atgaggtcat gcccccgcag gccctgagtg agcagactga ggaagcaggg gccgcactta301 gctccctccc cgaggtgcct ccctggactg gggaggtcaa cccacctctg agagacggca361 gtgctctagg gcgcagcccc tgggacccct ccgatagatc tgccctcttg aaaagcaaac421 tgagggctct gctcgctggc cctcggagcc tacgaagatc cagctgcttc gggggtagga481 ttgacaggat tggagcccag agtggactag gctgcaacag cttccggtac cgaagataac541 agccaaggag gaaaaggcag tcgattctgc ttgagcagat cgcaaaagat cctaagccct601 tgtggtgtgt cacgcagctt ggtcacattg ccactgtggc gtggtgaaca ccctcctgga661 gctgcggctt cctgccttca tctatcacga tcgatgttaa atgtagatga gtggtctagt721 ggggtcttgc ctctcccact ctgcatatta aggtagatcc tcaccctttt cagaaagcag781 ttggaaaaaa aaaaaaagaa taaacttcag caccaaggac agacgccgag gccctgatgt841 gcttctttgg cttctgccct cagttctttg ctctcccc

-   SEQ ID NO:18 is the nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 5:-   ggc agc ccc tgg gac ccc tcc gat aga tct gcc ctc ttg aaa ctg agg gct    ctg ctc gct ggc cct cgg agc cta cga aga tcc-   SEQ ID NO:19 is the nucleotide sequence encoding the amino acid    sequence of SEQ ID NO: 6:-   gtg tcc aac aca gat ctg atg gat ttc aag aac ctg cta gac cac ctg gag    gag aag atg ccg gta gaa gat gag gtc atg ccc ccg cag gcc ctg agt gag    cag act gag

DETAILED DISCLOSURE

The present invention pertains to a method for treating allergen-inducedairway reactivity by administering a natriuretic hormone peptide (NHP),or a nucleic acid sequence encoding an NHP, to a patient in needthereof, thereby ameliorating airway hyper-reactivity and/or airwayinflammation, which are characteristic of respiratory allergic disease,such as allergic rhinitis and asthma.

In specific embodiments, the peptides used in the subject inventioncomprise at least one amino acid sequence selected from the groupconsisting of NHP₁₋₃₀, NHP₃₁₋₆₇, NHP₇₉₋₉₈, and NHP₇₃₋₁₀₂, (SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5, respectively), SEQ ID NO:6,or a biologically active fragment or homolog thereof. In someembodiments, a combination of NHP or NHP-encoding nucleic acid sequencesis utilized. In one embodiment, the peptide utilized does not consist ofthe amino acid sequence of NHP₉₉₋₁₂₆ (SEQ ID NO:4). In otherembodiments, the peptides used in the subject invention comprise atleast one amino acid sequence selected from the group consisting of SEQID NO:7, and SEQ ID NO:16, or biologically active fragments or homologsof any of the foregoing.

According to the gene therapy method of the present invention, theNHP-encoding nucleic acid sequence is preferably administered to theairways of the patient, e.g., nose, sinus, throat and lung, for example,as nose drops, by nebulization, vaporization, or other methods known inthe art. More preferably, the nucleic acid sequence encoding NHP isadministered to the patient orally or intranasally, or otherwiseintratracheally. For example, the nucleic acid sequence can be inhaledby the patient through the oral or intranasal routes, or injecteddirectly into tracheal or bronchial tissue.

In specific embodiments, the nucleic acid sequences used in the subjectinvention encode at least one amino acid sequence selected from thegroup consisting of NHP₁₋₃₀, NHP₃₁₋₆₇, NHP₇₉₋₉₈, NHP₉₉₋₁₂₆, andNHP₇₃₋₁₀₂, (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQID NO:5, respectively), SEQ ID NO:6, or a biologically active fragmentor homolog of any of the foregoing. In other embodiments, the nucleicacid sequences used in the subject invention comprise at least onenucleotide sequence selected from the group consisting of SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17, or abiologically active fragment or homolog of any of the foregoing.

Preferably, the nucleic acid sequence encoding the NHP is administeredwith a nucleic acid sequence that is operatively linked with theNHP-encoding nucleic acid sequence and operates as a regulatorysequence. For example, the regulatory sequence can be a promotersequence that controls transcription and drives expression of theNHP-encoding nucleic acid sequence at the desired site, such as at, oradjacent to, the patient's respiratory epithelial cells. The promotercan be a constitutive or inducible promoter to allow selectivetranscription. The promoter can be a vertebrate or viral promoter.Optionally, enhancers may be used to obtain desired transcriptionlevels. An enhancer is generally any non-translated nucleic acidsequence that works contiguously with the coding sequence (in cis) tochange the basal transcription level dictated by the promoter.

The NHP-encoding nucleic acid sequences used in the methods, expressionvectors, and pharmaceutical compositions of the present invention arepreferably isolated. According to the present invention, an isolatednucleic acid molecule or nucleic acid sequence, is a nucleic acidmolecule or sequence that has been removed from its natural milieu. Assuch, “isolated” does not necessarily reflect the extent to which thenucleic acid molecule has been purified. An isolated nucleic acidmolecule or sequence useful in the present composition can include DNA,RNA, or any derivatives of either DNA or RNA. An isolated nucleic acidmolecule or sequence can be double stranded (i.e., containing both acoding strand and a complementary strand) or single stranded.

A nucleic acid molecule can be isolated from a natural source, or it canbe produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Nucleicacid molecules can be generated or modified using a variety oftechniques including, but not limited to, classic mutagenesis techniquesand recombinant DNA techniques, such as site-directed mutagenesis,chemical treatment of a nucleic acid molecule to induce mutations,restriction enzyme cleavage of a nucleic acid fragment, ligation ofnucleic acid fragments, polymerase chain reaction (PCR) amplificationand/or mutagenesis of selected regions of a nucleic acid sequence,synthesis of oligonucleotide mixtures and ligation of mixture groups to“build” a mixture of nucleic acid molecules, and combinations thereof.

Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably. As used herein, a“coding” nucleic acid sequence refers to a nucleic acid sequence thatencodes at least a portion of a peptide or protein (e.g., a portion ofan open reading frame), and can more particularly refer to a nucleicacid sequence encoding a peptide or protein which, when operativelylinked to a transcription control sequence (e.g., a promoter sequence),can express the peptide or protein.

The term “operably-linked” is used herein to refer to an arrangement offlanking sequences wherein the flanking sequences so described areconfigured or assembled so as to perform their usual function. Thus, aflanking sequence operably-linked to a coding sequence may be capable ofeffecting the replication, transcription and/or translation of thecoding sequence. For example, a coding sequence is operably-linked to apromoter when the promoter is capable of directing transcription of thatcoding sequence. A flanking sequence need not be contiguous with thecoding sequence, so long as it functions correctly. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter sequence and the coding sequence, and the promotersequence can still be considered “operably-linked” to the codingsequence. Each nucleotide sequence coding for NHP will typically haveits own operably-linked promoter sequence.

The nucleotide sequences encoding NHP used in the subject inventioninclude “homologous” or “modified” nucleotide sequences. Modifiednucleic acid sequences will be understood to mean any nucleotidesequence obtained by mutagenesis according to techniques well known topersons skilled in the art, and exhibiting modifications in relation tothe normal sequences. For example, mutations in the regulatory and/orpromoter sequences for the expression of a polypeptide that result in amodification of the level of expression of a polypeptide according tothe invention provide for a “modified nucleotide sequence”. Likewise,substitutions, deletions, or additions of nucleic acid to thepolynucleotides of the invention provide for “homologous” or “modified”nucleotide sequences. In various embodiments, “homologous” or “modified”nucleic acid sequences have substantially the same biological orserological activity as the native (naturally occurring) natriureticpeptide. A “homologous” or “modified” nucleotide sequence will also beunderstood to mean a splice variant of the polynucleotides of theinstant invention or any nucleotide sequence encoding a “modifiedpolypeptide” as defined below.

A homologous nucleotide sequence, for the purposes of the presentinvention, encompasses a nucleotide sequence having a percentageidentity with the bases of the nucleotide sequences of between at least(or at least about) 20.00% to 99.99% (inclusive). The aforementionedrange of percent identity is to be taken as including, and providingwritten description and support for, any fractional percentage, inintervals of 0.01%, between 20.00% and 99.99%. These percentages arepurely statistical and differences between two nucleic acid sequencescan be distributed randomly and over the entire sequence length.

In various embodiments, homologous sequences exhibiting a percentageidentity with the bases of the nucleotide sequences of the presentinvention can have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identitywith the polynucleotide sequences of the instant invention. Homologousnucleotide and amino acid sequences include mammalian homologs of thehuman NHP sequences.

The NHP homologs include peptides containing, as a primary amino acidsequence, all or part of an exemplified NHP polypeptide sequence. TheNHP homologs thus include NHP polypeptides having conservativesubstitutions, i.e., altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a peptide which is biologically active. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. In one aspect of thepresent invention, conservative substitutions for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs (see Table 1). Conservative substitutions alsoinclude substitutions by amino acids having chemically modified sidechains which do not eliminate the biological activity of the resultingNHP homolog.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Gly, Ser, Thr, Cys, Tyr, Asn, GinPolar Acidic Asp, Glu Basic Lys, Arg, His

Both protein and nucleic acid sequence homologies may be evaluated usingany of the variety of sequence comparison algorithms and programs knownin the art. Such algorithms and programs include, but are by no meanslimited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson andLipman Proc. Natl. Acad. Sci. USA, 1988, 85(8):2444-2448; Altschul etal. J. Mol. Biol., 1990, 215(3):403-410; Thompson et al. Nucleic AcidsRes., 1994, 22(2):46734680; Higgins et al. Methods Enzymol., 1996,266:383-402; Altschul et al. J. Mol. Biol., 1990, 215(3):403-410;Altschul et al. Nature Genetics, 1993, 3:266-272).

Identity and similarity of related nucleic acid molecules andpolypeptides can be readily calculated by known methods. Such methodsinclude, but are not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; York (1988); Biocomputing: Informatics and Genome Projects, Smith,D. W., ed., Academic Press, New York, 1993; York (1993); ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Jersey (1994); Sequence Analysisin Molecular Biology, von Heinje, G., Academic Press, 1987; Press(1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,M. Stockton Press, New York, 1991; York (1991); and Carillo et al., SLAMJ. Applied Math., 48:1073 (1988).

The methods, pharmaceutical compositions, and vectors of the presentinvention can utilize biologically active fragments of nucleic acidsequences encoding the 126-amino acid atrial natriuretic factor (ANF)prohormone, such as nucleic acid sequences encoding NHP₁₋₃₀, NHP₃₁₋₆₇,NHP₇₉₋₉₈, NHP₉₉₋₁₂₆, and NHP₇₃₋₁₀₂, (SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively), SEQ ID NO:6, andincluding biologically active fragments of the nucleic acid sequencesencoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6.

Representative fragments of the nucleotide sequences according to theinvention will be understood to mean any polynucleotide fragment havingat least 8 or 9 consecutive nucleotides, preferably at least 12consecutive nucleotides, and still more preferably at least 15 or atleast 20 consecutive nucleotides of the sequence from which it isderived. The upper limit for such fragments is one nucleotide less thanthe total number of nucleotides found in the full-length sequence (or,in certain embodiments, of the full length open reading frame (ORF)identified herein).

In other embodiments, fragments can comprise consecutive nucleotides of8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, and up to one nucleotide less than the full length ANF prohormone.In some embodiments, fragments comprise biologically active fragments ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQID NO:6.

It is also well known in the art that restriction enzymes can be used toobtain biologically active fragments of the nucleic acid sequences, suchas those encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, and SEQ ID NO:6. For example, Bal31 exonuclease can beconveniently used for time-controlled limited digestion of DNA (commonlyreferred to as “erase-a-base” procedures). See, for example, Maniatis etal. [1982] Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York; Wei et al. [1983] J Biol. Chem. 258:13006-13512.

The methods and pharmaceutical compositions of the present invention canutilize amino acid sequences that are biologically active fragments ofthe 126-amino acid atrial natriuretic factor (ANF) prohormone, such asNHP₁₋₃₀, NHP₃₁₋₆₇, NHP₇₉₋₉₈, NHP₉₉₋₁₂₆, and NHP₇₃₋₁₀₂ (SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively), SEQID NO:6, and including biologically active fragments of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

Representative fragments of the polypeptides according to the inventionwill be understood to mean any polypeptide fragment having at least 8 or9 consecutive amino acids, preferably at least 12 amino acids, and stillmore preferably at least 15 or at least 20 consecutive amino acids ofthe polypeptide sequence from which it is derived. The upper limit forsuch fragments is one amino acid less than the total number of aminoacids found in the full-length sequence.

In other embodiments, fragments of the polypeptides can compriseconsecutive amino acids of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, and up to one amino acid less than the full-length ANFprohormone. Fragments of polypeptides can be any portion of thefull-length ANF prohormone amino acid sequence (including human ornon-human mammalian homologs of the ANF prohormone) that exhibitbiological activity, e.g. a C-terminally or N-terminally truncatedversion of the ANF prohormone, or an intervening portion of the ANFprohormone. In some embodiments, fragments comprise biologically activefragments of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6.

The present invention can be practiced using other biologicallyequivalent forms of ANF fragments or homologs thereof as can beappreciated by the sequence comparison below. Sequence similaritiesbetween mouse and human forms of ANP are shown where areas ofconservation are clearly seen.

NCBI BLAST Comparison of mouse (Query) to human (Sbjct) ANP a.a.sequences. Query: 1MGSFSIT-LGFFLVLAFWLPGHIGANPVYSAVSNTDLMDFKMLLDHLEEKMPVEDEVMPP M SFS T + FL+LAF L G   ANP+Y+AVSN DLMDFKMLLDHLEEKMP+EDEV+PP Sbjct: 1MSSFSTTTVSFLLLLAFQLLGQTRANPMYNAVSNADLMDFKNLLDHLEEKMPLEDEVVPP Query: 60QALSEQTEEAGAALSSLPEVPPWTGEVNPPLRDGSALGRSPWDPSDXXXXXXXXXXXXXX Q LSE EEAGAALS LPEVPPWTGEV+P  RDG ALGR PWD SD Sbjct: 61QVLSEPNEEAGAALSPLPEVPPWTGEVSPAQRDGGALGRGPWDSSDRSALLKSKLRALLT Query: 120GPRSLRRSSCFGGRIDRIGAQSGLGCNSFRY 150  PRSLRRSSCFGGR+DRIGAQSGLGCNSFRYSbjct: 121 APRSLRRSSCFGGRMDRIGAQSGLGCNSFRY 151

The NHP of the invention can be prepared by well-known syntheticprocedures. For example, the polypeptides can be prepared by thewell-known Merrifield solid support method. See Merrifield (1963) JAmer. Chem. Soc. 85:2149-2154 and Merrifield (1965) Science 150:178-185.This procedure, using many of the same chemical reactions and blockinggroups of classical peptide synthesis, provides a growing peptide chainanchored by its carboxyl terminus to a solid support, usuallycross-linked polystyrene or styrenedivinylbenzene copolymer. This methodconveniently simplifies the number of procedural manipulations sinceremoval of the excess reagents at each step is effected simply bywashing of the polymer.

Alternatively, these peptides can be prepared by use of well-knownmolecular biology procedures. Polynucleotides, such as DNA sequences,encoding the NHP of the invention can be readily synthesized. Suchpolynucleotides are a further aspect of the present invention. Thesepolynucleotides can be used to genetically engineer eukaryotic orprokaryotic cells, for example, bacteria cells, insect cells, algaecells, plant cells, mammalian cells, yeast cells or fimgi cells forsynthesis of the peptides of the invention.

For purposes of the present invention, the biological activityattributable to the homologs and fragments of NHP and NHP-encodingnucleic acid sequences means the capability to prevent or alleviatesymptoms associated with allergic disease, such as bronchoconstrictionand inflammation. This biological activity can be mediated by one ormore of the following mechanisms: increased production of intracellularCa⁺⁺ concentration (e.g., in epithelial cells), increased production ofnitric oxide (NO), and decreased activation of NFkB.

The methods of the subject invention also contemplate the administrationof cells that have been genetically modified to produce NHP, orbiologically active fragments thereof. Such genetically modified cellscan be administered alone or in combinations with different types ofcells. Thus, genetically modified cells of the invention can beco-administered with other cells, which can include genetically modifiedcells or non-genetically modified cells. Genetically modified cells mayserve to support the survival and function of the co-administered cells,for example.

The term “genetic modification” as used herein refers to the stable ortransient alteration of the genotype of a cell of the subject inventionby intentional introduction of exogenous nucleic acids by any meansknown in the art (including for example, direct transmission of apolynucleotide sequence from a cell or virus particle, transmission ofinfective virus particles, and transmission by any knownpolynucleotide-bearing substance) resulting in a permanent or temporaryalteration of genotype. The nucleic acids may be synthetic, or naturallyderived, and may contain genes, portions of genes, or other usefulpolynucleotides in addition to those encoding NHP. A translationinitiation codon can be inserted as necessary, making methionine thefirst amino acid in the sequence. The term “genetic modification” is notintended to include naturally occurring alterations such as that whichoccurs through natural viral activity, natural genetic recombination, orthe like. The genetic modification may confer the ability to produceNHP, wherein the cell did not previously have the capability, or themodification may increase the amount of NHP produced by the cell, e.g.,through increased expression.

Exogenous nucleic acids and/or vectors encoding NHP can be introducedinto a cell by viral vectors (retrovirus, modified herpes virus, herpesvirus, adenovirus, adeno-associated virus, lentivirus, and the like) ordirect DNA transfection (lipofection, chitosan-nanoparticle mediatedtransfection, calcium phosphate transfection, DEAE-dextran,electroporation, and the like), microinjection, cationic lipid-mediatedtransfection, transduction, scrape loading, ballistic introduction andinfection (see, for example, Sambrook et al. [1989] Molecular Cloning: ALaboratory Manual, 2^(nd) Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

Preferably, the exogenous nucleic acid sequence encoding NHP is operablylinked to a promoter sequence that permits expression of the nucleicacid sequence in a desired tissue within the patient. The promoters canbe inducible or tissue specific as necessary.

The genetically modified cell may be chosen from eukaryotic orprokaryotic systems, for example bacterial cells (Gram negative or Grampositive), yeast cells, animal cells, plant cells, and/or insect cellsusing baculovirus vectors. In some embodiments, the genetically modifiedcell for expression of the nucleic acid sequences encoding NHP, arehuman or non-human mammal cells.

According to the methods of the present invention, NHP or nucleic acidsequences encoding NHP can be administered to a patient in order toalleviate (e.g., reduce or eliminate) a variety of symptoms associatedwith allergic diseases, in various stages of pathological development,such as airway reactivity, airway inflammation, and airway remodeling.Treatment with NHP or nucleic acid sequences encoding NHP is intended toinclude prophylactic intervention to prevent onset of the symptomsassociated with airway hyper-reactivity, airway inflammation, and airwayremodeling. The nucleic acid sequences and pharmaceutical compositionsof the invention can be co-admistered (concurrently or consecutively) toa patient with other therapeutic agents useful for treating airwayreactivity, airway inflammation, and airway remodeling.

Expression vectors for NHP are any which are known in the art that willcause expression of NHP-encoding nucleic acid sequences in mammaliancells. Suitable promoters and other regulatory sequences can be selectedas is desirable for a particular application. The promoters can beinducible or tissue specific as necessary. For example thecytomegalovirus (CMV) promoter (Boshart et al., Cell, 1985, 41:521-530)and SV40 promoter (Subramani et al., Mol. Cell. Biol., 1981, 1:854-864)have been found to be suitable, but others can be used as well.Optionally, the NHP-encoding nucleic acid sequences used in the subjectinvention include a sequence encoding a signal peptide upstream of theNHP-encoding sequence, thereby permitting secretion of the NHP from ahost cell. Also, various promoters may be used to limit the expressionof the peptide in specific cells or tissues, such as lung cells.

The pharmaceutical composition of the present invention can include aliposome component. According to the present invention, a liposomecomprises a lipid composition that is capable of fusing with the plasmamembrane of a cell, thereby allowing the liposome to deliver a nucleicacid molecule and/or a protein composition into a cell. Some preferredliposomes of the present invention include those liposomes commonly usedin, for example, gene delivery methods known to those of skill in theart. Some preferred liposome delivery vehicles comprise multilamellarvesicle (MLV) lipids and extruded lipids, although the invention is notlimited to such liposomes. Methods for preparation of MLV's are wellknown in the art. According to the present invention, “extruded lipids”are lipids which are prepared similarly to MLV lipids, but which aresubsequently extruded through filters of decreasing size, as describedin Templeton et al., Nature Biotech., 1997, 15:647-652, which isincorporated herein by reference in its entirety. Small unilarnellarvesicle (SUV) lipids can also be used in the composition and method ofthe present invention. Other preferred liposome delivery vehiclescomprise liposomes having a polycationic lipid composition (i.e.,cationic liposomes). For example, cationic liposome compositionsinclude, but are not limited to, any cationic liposome complexed withcholesterol, and without limitation, include DOTMA and cholesterol,DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.Liposomes of the present invention can be any size, including from about10 to 1000 nanometers (mn), or any size in between.

A liposome delivery vehicle of the present invention can be modified totarget a particular site in a mammal, thereby targeting and making useof a nucleic acid molecule of the present invention at that site.Suitable modifications include manipulating the chemical formula of thelipid portion of the delivery vehicle. Manipulating the chemical formulaof the lipid portion of the delivery vehicle can elicit theextracellular or intracellular targeting of the delivery vehicle. Forexample, a chemical can be added to the lipid formula of a liposome thatalters the charge of the lipid bilayer of the liposome so that theliposome fuses with particular cells having particular chargecharacteristics. In one embodiment, other targeting mechanisms, such astargeting by addition of exogenous targeting molecules to a liposome(i.e., antibodies) may not be a necessary component of the liposome ofthe present invention, since effective immune activation atimmunologically active organs can already be provided by the compositionwhen the route of delivery is intravenous or intraperitoneal, withoutthe aid of additional targeting mechanisms. However, in someembodiments, a liposome can be directed to a particular target cell ortissue by using a targeting agent, such as an antibody, soluble receptoror ligand, incorporated with the liposome, to target a particular cellor tissue to which the targeting molecule can bind. Targeting liposomesare described, for example, in Ho et al., Biochemistry, 1986, 25:5500-6; Ho et al., J Biol Chem, 1987a, 262: 13979-84; Ho et al., J BiolChem, 1987b, 262: 13973-8; and U.S. Pat. No. 4,957,735 to Huang et al.,each of which is incorporated herein by reference in its entirety). Inone embodiment, if avoidance of the efficient uptake of injectedliposomes by reticuloendothelial system cells due to opsonization ofliposomes by plasma proteins or other factors is desired, hydrophiliclipids, such as gangliosides (Allen et al., FEBS Lett, 1987, 223: 42-6)or polyethylene glycol (PEG)-derived lipids (Klibanov et al., FEBS Lett,1990, 268: 235-7), can be incorporated into the bilayer of aconventional liposome to form the so-called sterically-stabilized or“stealth” liposomes (Woodle et al., Biochim Biophys Acta, 1992, 1113:171-99). Variations of such liposomes are described, for example, inU.S. Pat. No. 5,705,187 to Unger et al., U.S. Pat. No. 5,820,873 to Choiet al., U.S. Pat. No. 5,817,856 to Tirosh et al.; U.S. Pat. No.5,686,101 to Tagawa et al.; U.S. Pat. No. 5,043,164 to Huang et al., andU.S. Pat. No. 5,013,556 to Woodle et al., all of which are incorporatedherein by reference in their entireties).

The NHP-encoding nucleic acid sequences of the present invention can beconjugated with chitosan. For example, DNA chitosan nanospheres can begenerated, as described by Roy, K et al. (1999, Nat Med 5:387). Chitosanallows increased bioavailability of the NHP-encoding nucleic acidsequences because of protection from degradation by serum nucleases inthe matrix and thus has great potential as a mucosal gene deliverysystem. Chitosan also has many beneficial effects, includinganticoagulant activity, wound-healing properties, and immunostimulatoryactivity, and is capable of modulating immunity of the mucosa andbronchus-associated lymphoid tissue.

Mammalian species which benefit from the disclosed methods of treatmentinclude, and are not limited to, apes, chimpanzees, orangutans, humans,monkeys; domesticated animals (e.g., pets) such as dogs, cats, guineapigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. The term“patient” is intended to include such human and non-human mammalianspecies. According to the method of the present invention, human ornon-human mammalian NHP (or nucleic acid sequences encoding human ornon-human mammalian NHP) can be administered to the patient. The NHP maybe naturally occuring within the patient's species or a differentmammalian species. The expression vectors used in the subject inventioncan comprise nucleic acid sequences encoding any human or non-humanmammalian NHP.

In another aspect, the present invention concerns pharmaceuticalcompositions containing a therapeutically effective amount of NHP, ornucleic acid sequences encoding NHP, and a pharmaceutically acceptablecarrier. Preferably, the NHP-encoding nucleic acid sequences arecontained within an expression vector, such as plasmid DNA or virus. Thepharmaceutical composition can be adapted for administration to theairways of the patient, e.g., nose, sinus, throat and lung, for example,as nose drops, as nasal drops, by nebulization as an inhalant,vaporization, or other methods known in the art. Administration can becontinuous or at distinct intervals as can be determined by a personskilled in the art.

The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Furthermore, as used herein, the phrase“pharmaceutically acceptable carrier” means any of the standardpharmaceutically acceptable carriers. The pharmaceutically acceptablecarrier can include diluents, adjuvants, and vehicles, as well asimplant carriers, and inert, non-toxic solid or liquid fillers,diluents, or encapsulating material that does not react with the activeingredients of the invention. Examples include, but are not limited to,phosphate buffered saline, physiological saline, water, and emulsions,such as oil/water emulsions. The carrier can be a solvent or dispersingmedium containing, for example, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils. Formulations containingpharmaceutically acceptable carriers are described in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Sciences (Martin E W[1995] Easton Pa., Mack Publishing Company, 19th ed.) describesformulations that can be used in connection with the subject invention.Formulations suitable for parenteral administration include, forexample, aqueous sterile injection solutions, which may containantioxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and nonaqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the condition of the sterile liquid carrier, for example,water for injections, prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powder, granules, tablets,etc. It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of the subject inventioncan include other agents conventional in the art having regard to thetype of formulation in question.

The NHP or nucleic acid sequences encoding NHP (and pharmaceuticalcompositions containing them) can be administered to a patient by anyroute that results in prevention or alleviation of symptoms associatedwith allergic disease, such as bronchoconstriction and/or inflammation.For example, the NHP or NHP-encoding nucleic acid molecule can beadministered parenterally, intravenously (I.V.), intramuscularly (I.M.),subcutaneously (S.C.), intradermally (I.D.), orally, intranasally, etc.Examples of intranasal administration can be by means of a spray, drops,powder or gel and also described in U.S. Pat. No. 6,489,306, which isincorporated herein by reference in its entirety. One embodiment of thepresent invention is the administration of the invention as a nasalspray. Alternate embodiments include administration through any oral ormucosal routes, sublingual administration and even eye drops. However,other means of drug administrations are well within the scope of thepresent invention.

The NHP or NHP-encoding nucleic acid molecule is administered and dosedin accordance with good medical practice, taking into account theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight, and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. For example,an effect amount of NHP-encoding nucleic acid molecule is that amountnecessary to provide a therapeutically effective amount of NHP, whenexpressed in vivo. The amount of NHP or NHP-encoding nucleic acidmolecule must be effective to achieve improvement including but notlimited to total prevention and to improved survival rate or more rapidrecovery, or improvement or elimination of symptoms associated withallergen-induced airway hyper-reactivity and other indicators as areselected as appropriate measures by those skilled in the art Inaccordance with the present invention, a suitable single dose size is adose that is capable of preventing or alleviating (reducing oreliminating) a symptom in a patient when administered one or more timesover a suitable time period. One of skill in the art can readilydetermine appropriate single dose sizes for systemic administrationbased on the size of a mammal and the route of administration.

Standard molecular biology techniques known in the art and notspecifically described were generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and in Watson et al., Recombinant DNA, Scientific AmericanBooks, New York and in Birren et al. (eds) Genome Analysis: A LaboratoryManual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York(1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828;4,683,202; 4,801,531; 5,192,659; and 5,272,057; and incorporated hereinby reference. Polymerase chain reaction (PCR) was carried out generallyas in PCR Protocols: A Guide To Methods And Applications, AcademicPress, San Diego, Calif. (1990). In situ (In-cell) PCR in combinationwith Flow Cytometry can be used for detection of cells containingspecific DNA and mRNA sequences (Testoni et al., Blood, 1996, 87:3822.)

The term “gene therapy”, as used herein, refers to the transfer ofgenetic material (e.g., DNA or RNA) of interest into a host to treat orprevent a genetic or acquired disease or condition phenotype. Thegenetic material of interest encodes a product (e.g. a protein,polypeptide, peptide, or functional RNA) whose production in vivo isdesired. For example, the genetic material of interest can encode ahormone, receptor, enzyme, polypeptide or peptide of therapeutic value.For a review see, in general, the text “Gene Therapy” (Advances inPharmacology 40, Academic Press, 1997).

Two basic approaches to gene therapy have evolved: (1) ex vivo and (2)in vivo gene therapy. In ex vivo gene therapy, cells are removed from apatient and, while being cultured, are treated in vitro. Generally, afunctional replacement gene is introduced into the cell via anappropriate gene delivery vehicle/method (transfection, transduction,homologous recombination, etc.) and an expression system as needed andthen the modified cells are expanded in culture and returned to thehost/patient. These genetically reimplanted cells have been shown toproduce the transfected gene product in situ.

In in vivo gene therapy, target cells are not removed from the subject,rather the gene to be transferred is introduced into the cells of therecipient organism in situ, that is within the recipient. Alternatively,if the host gene is defective, the gene is repaired in situ. Thesegenetically altered cells have been shown to produce the transfectedgene product in situ.

The gene expression vector is capable of delivery/transfer ofheterologous nucleic acid sequences into a host cell. The expressionvector may include elements to control targeting, expression andtranscription of the nucleic acid sequence in a cell selective manner asis known in the art. It should be noted that often the 5′UTR and/or3′UTR of the gene may be replaced by the 5′UTR and/or 3′UTR of theexpression vehicle.

The expression vector can include a promoter for controllingtranscription of the heterologous material and can be either aconstitutive or inducible promoter to allow selective transcription. Theexpression vector can also include a selection gene.

Vectors can be introduced into cells or tissues by any one of a varietyof known methods within the art. Such methods can be found generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press,Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, AnnArbor, Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and include, for example,stable or transient transfection, lipofection, electroporation, andinfection with recombinant viral vectors.

Introduction of nucleic acids by infection offers several advantagesover the other listed methods. Higher efficiency can be obtained due totheir infectious nature. Moreover, viruses are very specialized andtypically infect and propagate in specific cell types. Thus, theirnatural specificity can be used to target the vectors to specific celltypes in vivo or within a tissue or mixed culture of cells. Viralvectors can also be modified with specific receptors or ligands to altertarget specificity through receptor mediated events.

A specific example of a DNA viral vector for introducing and expressingrecombinant sequences is the adenovirus derived vector Adenop53TK. Thisvector expresses a herpes virus thymidine kinase (TK) gene for eitherpositive or negative selection and an expression cassette for desiredrecombinant sequences. This vector can be used to infect cells that havean adenovirus receptor which includes most cancers of epithelial originas well as others. This vector as well as others that exhibit similardesired functions can be used to treat a mixed population of cells andcan include, for example, an in vitro or ex vivo culture of cells, atissue or a human subject.

Additional features can be added to the vector to ensure its safetyand/or enhance its therapeutic efficacy. Such features include, forexample, markers that can be used to negatively select against cellsinfected with the recombinant virus. An example of such a negativeselection marker is the TK gene described above that confers sensitivityto the antibiotic gancyclovir. Negative selection is therefore a meansby which infection can be controlled because it provides induciblesuicide through the addition of antibiotic. Such protection ensures thatif, for example, mutations arise that produce altered forms of the viralvector or recombinant sequence, cellular transformation will not occur.Features that limit expression to particular cell types can also beincluded. Such features include, for example, promoter and regulatoryelements that are specific for the desired cell type.

In addition, recombinant viral vectors are useful for in vivo expressionof a desired nucleic acid because they offer advantages such as lateralinfection and targeting specificity. Lateral infection is inherent inthe life cycle of, for example, retrovirus and is the process by which asingle infected cell produces many progeny virions that bud off andinfect neighboring cells. The result is that a large area becomesrapidly infected, most of which was not initially infected by theoriginal viral particles. This is in contrast to vertical-type ofinfection in which the infectious agent spreads only through daughterprogeny. Viral vectors can also be produced that are unable to spreadlaterally. This characteristic can be useful if the desired purpose isto introduce a specified gene into only a localized number of targetedcells.

In another aspect, the present invention concerns an isolated peptidecomprising the amino acid sequence NHP₇₃₋₁₀₂ (SEQ ID NO:5), or abiologically active fragment or homolog thereof. NHP₇₃₋₁₀₂ is aminoacids 73-102 of the 151-amino acid long human atrial natriuretic factor(ANF)). In another aspect, the present invention concerns an isolatedpeptide comprising the amino acid sequence of SEQ ID NO:6, or abiologically active fragment or homolog thereof. SEQ ID NO:6 is abiologically active fragment of the human ANF. In another aspect, thepresent invention concerns an isolated nucleic acid molecule encodingthe amino acid sequence of NHP₇₃₋₁₀₂ (SEQ ID NO:5), or a biologicallyactive fragment or homolog thereof. In another aspect, the presentinvention concerns an isolated nucleic acid molecule (SEQ ID NO:13)encoding the amino acid sequence of SEQ ID NO:6, or a biologicallyactive fragment or homolog thereof.

As used herein, the terms “peptide”, “polypeptide”, and “protein” referto amino acid sequences of any length unless otherwise specified.

EXAMPLE 1 Expression of NHP in Human Type-II Alveolar A549 Cells andMurine Lung

A. Materials and Methods

Animals. Six-week old female BALB/c mice from Jackson laboratory (BarHarbor, Me.) were maintained in pathogen free conditions in accordancewith animal research committee regulations.

Construction of NHP expression vector. Total RNA was isolated frommurine heart using Trizol reagent (LIFE TECHNOLOGY, Gaithersburg, Md.)following the manufacturer's protocol. The cDNA sequence for the 151amino acid long pre-pro hormone ANF was amplified by RT-PCR. SEQ IDNO:13 was amplified using primers listed in SEQ ID NO:8 and SEQ ID NO:9.NHP₇₃₋₁₀₂ was amplified using primers listed in SEQ ID NO:10 and SEQ IDNO:11. A translation initiation codon was inserted in the forwardprimers (SEQ ID NO:8 and SEQ ID NO:10), so that the recombinant peptideshad an additional amino acid, methionine, as the first amino acid apartfrom its known amino acid content. The PCR product was cloned in pVAXvector (INVITROGEN, Carlsbad, Calif.) at HindIII and XhoI sites. Thecloned NHP₇₃₋₁₀₂ sequence was verified by DNA sequencing and itsexpression was checked in A549 human epithelial cells.

Estimation of NHP by EIA. The expression of NHP was measured byutilizing a commercial kit (SPI-BIO, France) according to themanufacturer's instructions. The kit measures the rat NHP, which ishomologous to the mouse NHP used in the present study.

B. Results

NHP was amplified using PCR and the product was cloned into pVAX vector,as described. Either pNHP₇₃₋₁₀₂ or empty plasmid vector pVAX weretransfected to the human type-II alveolar cells (A549). To confirm theexpression of NHP, cells and supernatants were collected 48 hourspost-transfection and NHP concentrations were measured by ELISA. Cellstransfected with pNHP produced NHP in both supernatant and cell extracts(FIG. 2A), whereas the cells transfected with pVAX did not. Theseresults show that the NHP₇₃₋₁₀₂ peptide was expressed and secreted intothe culture medium from the A549 cells.

EXAMPLE 2 pNHP Delivered with Lipofectin Intranasally Attenuates AirwayReactivity in Ovalbumin-Sensitized Mice

A. Materials and Methods

Animals. Six-week old female BALB/c mice from Jackson laboratory (BarHarbor, Me.) were maintained in pathogen free conditions in accordancewith animal research committee regulations.

Allergen Sensitization, Intranasal Gene Transfer. BALB/c mice (n=6) weresensitized once with intraperitoneal (i.p.) injection of 10 μg ovalbumin(OVA) precipitated with 1 mg of alum on day 1. Mice were intranasaly(i.n.) administered three times a day with either 25 μg/mouse pNHP₇₃₋₁₀₂or vector control (pVAX) (10 μg of lipofectamine in PBS) on days 15, 18and 21, as shown in FIG. 3A.

RT-PCR analysis. Total RNA was isolated from murine lung and spleentissue using Trizol reagent (LIFE TECHNOLOGY, Gaithersburg, Md.) andRT-PCR was performed utilizing ANP specific primers as described before(Kumar M. et al., Vaccine 1999; 18:558-567). The resultant PCR productswere analyzed by electrophoresis on a 1.5% agarose gel and the productsvisualized by staining with ethidium bromide.

Measurement of Ova specific IgE. Ova specific IgE was measured tomonitor the degree of sensitization. Microtitre plates were coatedovernight at 4° C. with 100 μl of OVA (5 μg/ml). Sera obtained at day 21from sensitized and non-sensitized mice (n=4) were incubated to theantigen-coated wells and bound IgE was detected with biotinylatedanti-mouse IgE (02112D; Pharmingen, Calif.). Biotin anti-mouse IgE(02122D) reacts specifically with the mouse IgE of Igh^(a) and Igh^(b)haplotypes and does not react with other IgG isotypes. Dilutedstreptavidin-peroxidase conjugate was added, the bound enzyme detectedwith TMB, and the absorbance read at 450 nm.

Pulmonary Function. Airway hyperreactivity was measured in animals threedays after the last intranasal DNA delivery following 24 hours ofchallenge with the ovalbumin (50 μg/mouse using a whole bodyplethysmograph (BUXCO, Troy, N.Y.), as previously described (Matsuse, H.et al., J. Immunol., 2000, 164:6583-6592). Allergen-induced airwayhyper-responsiveness (AHR) was measured on 10 days interval up to day56. Mice were ova challenged 24 hours prior to each AHR measurement.

Statistical analysis. Pairs of groups were compared by the student'st-test. Differences between groups were considered significant atp<0.05. Values for all measurements are expressed as the mean±SD.

B. Results

The effect of pNHP gene transfer was examined in an ovalbumin-sensitizedBALB/c mouse model. RT-PCR was performed to confirm the expression ofANP in murine lung and spleen. ANP specific transcript was observed inthe lung tissue of mice receiving pANP construct and not from the micereceiving the empty plasmid vector (FIG. 1B). No expression of ANP wasobserved from the spleen tissue (data not shown).

Ovalbumin-specific IgE was measured in the serum to determine the degreeof sensitization achieved following ovalbumin injection. Mice thatreceived ovalbumin with alum had significantly (p<0.01) higherovalbumin-specific IgE titers than the control group of mice thatreceived PBS (FIG. 3C).

AHR was measured in BALB/c mice, sensitized with ovalbumin andadministered with pNHP₇₃₋₁₀₂ or control vector plasmid prior toovalbumin challenge. The control mice received empty vector. Theexperimental outline is shown in FIG. 3A. Mice administered pNHP showedsignificantly less AHR (p<0.05) than did controls to inhaledmethacholine (FIG. 3D). Prophylactic treatment with either plasmid (SEQID NO:13) or pNHP₇₃₋₁₀₂ showed a reduction in % Penh suggesting thatboth plasmid are capable of prophylactically attenuating AHR.

To determine the length of protection, following pNHP gene transfer micewere challenged at 10 d interval. The protection lasted for over aperiod of 25 days during which the AHR of pNHP treated mice wassignificantly lower (P<0.05) than the mice receiving control plasmid(FIGS. 4A and B). Although there was a decrease in the AHR on day 56,the differences between values were not significant.

EXAMPLE 3 Chitosan-pNHP Nanoparticles Administered Intranasally DecreaseAirway Hyper-Reactivity and Inflammation

A. Materials and Methods

Animals. Female 6 to 8 week-old wild type BALB/c mice from JacksonLaboratory (Bar Harbor, Me.) were maintained in pathogen-free conditionsat the University of South Florida College of Medicine vivarium. Allprocedures were reviewed and approved by the committees on animalresearch at the University of South Florida College of Medicine and VAHospital.

Preparation of chitosan-pNHP nanoparticles. pNHP₇₃₋₁₀₂ encoding DNA wascloned in the mammalian expression vector pVAX (INVITROGEN, San Diego,Calif.), and complexed with chitosan, as described previously (Hulks, G.et al., Clin Sci, 1990, 79:51-55; Angus, R. M. et al., Clin Exp Allergy,1994, 24:784-788). Briefly, recombinant plasmid dissolved in 25 mMNa₂SO₄ was heated for 10 min at 55° C. Chitosan (VANSON, Redmond, Wash.)was dissolved in 25 mM Na acetate, pH 5.4, to a final concentration of0.02% and heated for 10 min at 55° C. After heating, chitosan and DNAwere mixed, vortexed vigorously for 20-30 seconds, and stored at roomtemperature until use.

Reversal of established AHR. Mice were sensitized i.p. with 50 μgOVA/alum on day 1 followed by intranasal challenge with 50 μg of OVA onday 14. On days 21-23, mice were given 25 μg of pNHP/chitosan i.n. permouse. Mice were further challenged i.n. with OVA (50 μg/mouse) on days27 through 29 and AHR was measured on day 30. Mice were bled andsacrificed on day 31, and spleens and lungs were removed.

Examination of bronchoalveolar lavage fluid (BALF). Mice were sacrificedand lungs were lavaged with 1 ml of PBS introduced through the trachea.The BALF was centrifuged 10 min. at 300×g and cells were rinsed with PBSand resuspended. Aliquots of the cell suspension were applied to slidesusing a CYTOSPIN apparatus (SHANDON SOUTHERN), stained and examinedmicroscopically. Cells were identified as monocytes, eosinophils,neutrophils, and lymphocytes by morphological characteristics. Twoslides per mouse (n=4) were counted by three blinded investigators.

Statistical analysis. Pairs of groups were compared by the student'st-test. Differences between groups were considered significant atp<0.05. Values for all measurements are expressed as the mean±SD.

B. Results

A combination of allergen exposure and respiratory syncytial virusinduces chronic asthma phenotype in BAALB/c mice. To determine whethertherapeutic administration of chitosan-pNHP can attenuate establishedasthma induced by allergen exposure and RSV infection, mice were firstsensitized and challenged with OVA and then infected with RSV andsubsequently given Chitosan+pNHP (CHIpP) therapy, as shown in theprotocol of FIG. 5A. Airway hyper-reactivity (%Penh) was measured bywhole body plethysmography (FIG. 5B). The results show a completereversal to the basal level of AHR in the group of mice that weretreated with CHIpP. To determine whether CHIpP therapy decreasesestablished pulmonary inflammation, lungs of treated and thenOVA-challenged mice were lavaged and BAL cells were examined. The numberof eosinophils in the BAL fluid showed a significant reduction in theCHIpP-treated mice compared with the untreated control group, as shownin FIG. 5C.

EXAMPLE 4 pNHP Induces Production of Nitric Oxide by ActivatingConstitutive Nitric Oxide Synthase in Human in Lung Epithelial Cells

A. Materials and Methods

Cell lines and culture conditions. The human alveolar type II epithelialcell line A549 (ATCC) was cultured at 37° C. in Dulbecco's modifiedEagle's medium containing 10% fetal bovine serum and 100 U/ml each ofpenicillin and streptomycin in an atmosphere of 5% CO2/95% air. Cellswere subcultured weekly and used between passages 9 and 22. Experimentswere repeated with primary NHBE obtained from CLONETICS (Walkersville,Md.) from pooled donors. These cells were cultured in BEBM mediumsupplied by the vendor and supplemented with 10% fetal bovine serum anda mix of growth factors without antibiotics. Cells were grown at 37° C.in 5% CO₂/95% and used between passages 3 and 9.

Expression plasmids and transfection. The construction of plasmidencoding NHP₇₃₋₁₀₂ has been descnbedpreviously (Kumar, M et al., JAllergy Clin Immunol., 2002, 110:879-82). For transfection of epithelialcells, cells at 60% confluence (log phase) were transfected 4 hr at 37°C. with plasmid DNA (1 μg per 10⁶ cells) complexed with lipofectamine(GIBCOBRL Life Technologies). Complete medium was then added to thecultures and the cells were incubated at 37° C. for 24 to 48 h to allowexpression of the natriuretic peptides.

Assay for nitric oxide. The assay for nitric oxide (NO) is based on thatof Misko et al. (Misko, T P et al., Anal Biochem., 1993, 214:11-6) andmeasures nitrite, the stable breakdown product of NO, which is reactedwith diaminonaphthalene to produce a fluorescent compound. A549 or NHBEcells were transfected with plasmid/lipofectamine complexes as describedabove. At specific time points, 100 μl samples of culture medium wereremoved and stored at −20° C. After all samples were taken, they werecleared by centrifugation and 10 μl of a freshly prepared solution of0.02 mg/ml diaminonaphthalene was added to each tube, shaken, andallowed to react for 10 min at room temperature. The reaction wasstopped by addition of 30 μl of 0.5 M NaOH and the fluorescence of thesamples was read using a quartz microcuvet (3 mm path length) in a JASCOspectrofluorometer with excitation at 365 mn and emission at 409 nm.Nitrite standards were run in the same medium as the experimentalsamples to generate a standard curve which was used to calibrate thereadings. As a positive control, one set of wells was incubated with 1μM calcium ionophore, A23187 (SIGMA).

B. Results

NO is a bronchodilator and Ca⁺⁺-calmodulin binding activates theconstitutive form of nitric oxide synthase (cNOS) in epithelial cells(Howarth, P. H. et al., Int Arch Allergy Immunol., 1995, 107:228-30). Todetermine whether the increased intracellular Ca⁺⁺ seen inNHP₇₃₋₁₀₂-transfected cells affects nitric oxide (NO) levels, aliquotsof the medium were removed before the Ca⁺⁺ assay and mixed withdiaminonaphthalene which reacts with nitrite (from the reaction of NOand water) to produce a fluorescent compound. NO generation wassignificantly higher in cells expressing NHP₇₃₋₁₀₂ (FIGS. 3A and B). Toverify that NO production was due to the constitutive NOS, one aliquotof cells was incubated during the expression phase with 1 mMN_(ω)-nitro-L-arginie methyl ester, an arginine analog that blocks cNOSproduction of NO. The enhanced NO generation was inhibited bypretreatment of the cells with N-nitro-L-arginine methyl ester, whichblocks cNOS activity (FIG. 3C). Airway smooth muscle hypertrophy andhyperplasia are inportant determinants of airway remodeling andbronchial responsiveness in asthma. NHP₇₃₋₁₀₂ appears to act onepithelial cells to produce NO via constitutive NOS, which in turncontrols bronchial hyperreactivity and proliferation of airway smoothmuscle cells.

EXAMPLE 5 pNHP Induces Anti-Inflammatory Response in the Lung byDecreasing NFkB Activation of Epithelial Cells

A. Materials and Methods

Luciferase reporter assay for NFκB activation. A549 and NHBE cells weregrown to about 60% confluence in 12-well culture plates and transfectedusing LIPOFECTAMINE 2000 (INVITROGEN, Carlsbad Calif.). Cells weretransfected with a luciferase construct under the control of anNFκB-activatable promoter (MERCURY PROFILING SYSTEM, CLONTECH, Palo AltoCalif.) and pLacZ as a normalization control either with or withoutpANP. Relative amounts of plasmid DNA and lipofectamine reagent wereoptimized for NHBE cells and cells were transfected for 4 h inserum-free DMEM without antibiotics at 37° C. After transfection, DMEMwith 10% FBS was added and cells were incubated for 24 to 48 h at 37° C.Cells were harvested at specific time points and lysates were assayedfor luciferase activity using the DUAL LUCIFERASE Assay System (PROMEGA,Madison Wis.) read in an MLX microplate luminometer (DYNEX TECHNOLOGIES,Chantilly Va.). Transfection efficiencies were normalized by measuringβ-galactosidase activity.

Statistical analysis. Experiments were repeated a minimum of three timesand data are expressed as means±SEM. Pairs of groups were comparedthrough the use of Student's t tests. Differences between groups wereconsidered significant at p≦0.05.

B. Results

To study the potential role of NHP in regulating inflammation, A549cells were transfected with pNHP plasmid encoding amino acids 73-102(pNHP₇₃₋₁₀₂) and the activation of NFkB, which controls a number ofgenes encoding proinflammatory molecules and is key to the inflammatorycascade and has been linked to inflammation, was examined (Ishii, Y. etal., J Anat., 1989, 166:85-95; Boiteau, R. et al., Am Rev Res Dis.,1988, 137:A484). The results showed that cells cotransfected with pNHPsignificantly decreased luciferase activity compared to control plasmidsuggesting that pNHP is capable of inhibiting PMA-induced NFκBactivation in A549 cells and in NHBE cells, as shown in FIGS. 6A-6B.These results indicate that pNHP possesses potential anti-inflammatoryactivities, and that it may exert its bronchodilatory effect bystimulating the production of NO and its anti-inflammatory effect bydeactivating NFκB, the central molecule controlling inflammation inasthmatic airways.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A pharmaceutical composition comprising: a nucleic acid sequence encoding a natriuretic hormone peptide comprising an amino acid sequence comprising SEQ ID NO:5 or a homolog of SEQ ID NO: 5 having at least one conservative amino acid substitution of SEQ ID NO: 5, and an operably linked promoter sequence; and a pharmaceutically acceptable carrier.
 2. An expression vector comprising: a nucleic acid sequence encoding a natriuretic hormone peptide comprising an amino acid sequence comprising SEQ ID NO: 5 or a homolog of SEQ ID NO: 5 having at least one conservative amino acid substitution of SEQ ID NO: 5, or comprising an amino acid sequence of SEQ ID NO: 6; and an operably linked promoter sequence.
 3. The expression vector of claim 2, wherein the natriuretic hormone peptide comprises an amino acid sequence comprising SEQ ID NO:
 6. 4. An isolated cell comprising a nucleic acid sequence encoding a natriuretic hormone peptide comprising an amino acid sequence comprising SEQ ID NO:5 or a homolog of SEQ ID NO: 5 having at least one conservative amino acid substitution of SEQ ID NO: 5, or comprising SEQ ID NO: 6; and an operably linked promoter sequence.
 5. The isolated cell of claim 4, wherein the natriuretic hormone peptide comprises an amino acid sequence comprising SEQ ID NO:
 6. 6. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence comprising SEQ ID NO: 5 or a homolog of SEQ ID NO: 5 having at least one conservative amino acid substitution of SEQ ID NO: 5, or comprising SEQ ID NO:
 6. 7. The pharmaceutical composition of claim 1, further comprising a chitosan.
 8. An expression vector comprising: a nucleic acid sequence encoding a natriuretic hormone peptide comprising an amino acid sequence comprising SEQ ID NO: 5 or a homolog of SEQ ID NO: 5 having at least one conservative amino acid substitution of SEQ ID NO: 5, and an operably linked promoter sequence.
 9. An isolated cell comprising a nucleic acid sequence encoding a natriuretic hormone peptide comprising an amino acid sequence comprising SEQ ID NO: 5 or a homolog of SEQ ID NO: 5 having at least one conservative amino acid substitution of SEQ ID NO: 5, and an operably linked promoter sequence.
 10. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a natriuretic hormone peptide comprising an amino acid sequence comprising SEQ ID NO: 5 or a homolog of SEQ ID NO:5 having at least one conservative amino acid substitution of SEQ ID NO:
 5. 11. The pharmaceutical composition of claim 1, further comprising a liposome.
 12. The expression vector of claim 8, wherein the expression vector is a DNA plasmid.
 13. The pharmaceutical composition according to claim 1, wherein the natriuretic hormone peptide consists of the amino acid sequence of SEQ ID NO:
 5. 14. The expression vector according to claim 8, wherein the natriuretic hormone peptide consists of the amino acid sequence of SEQ ID NO:
 5. 15. The isolated cell according to claim 9, wherein the natriuretic hormone peptide consists of the amino acid sequence of SEQ ID NO:
 5. 16. The isolated nucleic acid sequence according to claim 10, wherein the natriuretic hormone peptide consists of the amino acid sequence of SEQ ID NO:
 5. 17. The pharmaceutical composition according to claim 1, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:
 18. 18. The expression vector according to claim 2, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO:
 19. 19. The isolated cell according to claim 4, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO:
 19. 20. The isolated nucleic acid sequence according to claim 6, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO:
 19. 21. The expression vector according to claim 8, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:
 18. 22. The isolated cell according to claim 9, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:
 18. 23. The isolated nucleic acid sequence according to claim 10, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:
 18. 24. A pharmaceutical composition comprising: a nucleic acid molecule comprising a nucleic acid sequence encoding a natriuretic hormone peptide comprising an amino acid sequence comprising SEQ ID NO: 6, and an operably linked promoter sequence; and a pharmaceutically acceptable carrier.
 25. The pharmaceutical composition of claim 24, further comprising a liposome.
 26. The pharmaceutical composition according to claim 24, wherein the natriuretic hormone peptide consists of the amino acid sequence of SEQ ID NO:
 6. 27. The pharmaceutical composition according to claim 24, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:
 19. 28. The pharmaceutical composition according to claim 1, wherein the natriuretic hormone peptide comprises the amino acid sequence of SEQ ID NO:
 5. 29. The expression vector according to claim 8, wherein the natriuretic hormone peptide comprises the amino acid sequence of SEQ ID NO:
 5. 30. The isolated cell according to claim 9, wherein the natriuretic hormone peptide comprises the amino acid sequence of SEQ ID NO:
 5. 31. The isolated nucleic acid molecule according to claim 10, wherein the natriuretic hormone peptide comprises the amino acid sequence of SEQ ID NO:
 5. 32. The expression vector according to claim 2, wherein the natriuretic hormone peptide comprises an amino acid sequence consisting of SEQ ID NO:
 6. 33. The isolated cell according to claim 4, wherein the natriuretic hormone peptide comprises an amino acid sequence consisting of SEQ ID NO:
 6. 34. The isolated nucleic acid sequence according to claim 6, wherein the amino acid sequence consists of SEQ ID NO:
 6. 